The endogenous phosphorylation of synaptic plasma membrane (SPM) and synaptosome (SYN) fractions of mouse brain was assayed in vitro using AT32P and 32Pi as phosphate donors, respectively. Specific protein phosphorylation was analyzed by autoradiography following SDS polyacrylamide gel electrophoresis. Addition of 50-300 microns Ca ions or heated SYN cytosol caused a concentration related increase in the phosphorylation of SPM proteins with apparent molecular weights of 15, 52, 59, and 89 K daltons. The phosphorylation of SYN proteins with apparent molecular weights of 30, 46, 78 and 89 K daltons was increased when this fraction was incubated in the presence of Ca ions or Ca ions plus the Ca ions ionophore, A23187. The effects of Ca ions, heated cytosol and A23187 on specific protein phosphorylation were markedly enhanced when the SPM and SYN fraction were obtained from mice rendered tolerant to and dependent on morphine. In contrast, the phosphorylation of the same proteins was reduced when the SPM and SYN fractions were derived from mice undergoing naloxone-precipitated withdrawal.